Information flow within the nervous system requires the perpetuation of ionic gradients along neurons. In many neurons, effective and efficient perpetuation of such gradients along axons requires electrical insulation. Myelin, a lipid-rich, dielectric substance that ensheathes axons, serves this insulating function. The nervous system contains high levels of myelin, which is especially enriched where many myelinated axons are bundled together, such as in tracts of the spinal cord and spinal nerve roots, nerves in the peripheral nervous system, and fiber tracts in the brain, collectively called “white matter”, as opposed to “grey matter”. Because non-nervous system tissue lacks myelin, the presence of myelin can distinguish nerve tissue from other tissue types; the spinal cord and spinal nerve roots from non-nervous elements of the vertebral column, and white matter from grey matter in the brain.
The ability to qualitatively or quantitatively visualize myelin, either in vivo or in vitro, confers upon researchers and clinicians important diagnostic and treatment tools. For example, the ability to visually identify peripheral nerves during surgery assists surgeons in avoiding cutting or damaging nerves. Previous efforts in image-guided surgery of nerves utilized modalities that would not require contrast agents or fluorescent labeling of axons by retrograde transport. A challenge in the first approach is that the signal is typically ambiguous
Retrograde labeling of nerves in animal models is widely reported in the literature. Although this strategy may work, there are many inherent problems. Labeling would depend on exactly where the contrast agent is injected. If the nerves fail to take up the contrast agent, the nerve would not be visualized. In some cases, nerve stimulation is required to facilitate retrograde transport. The long times required for retrograde transport may not be clinically feasible.
Myelinated nerves and fiber tracts serve as useful landmarks in anatomical studies carried out by preclinical and basic neuroscience researchers. Furthermore, the formation of myelin sheaths is an important step in the generation and functional stability of new neurons; so the availability of myelin markers may aid researchers study such processes. Myelin-labeling methodologies are also useful in the development of numerous therapies, neural stem cell research, and putative animal models of myelin-associated neuropathies. In vivo myelin imaging of the spinal cord assists clinicians in the diagnosis and treatment of spinal cord pathology, such as nerve compression or herniated discs as well as myelin-associated neuropathies, such as multiple sclerosis which results in damage to myelin within the central or peripheral nervous system. The ability to measure amounts of myelination in vivo in patients with such conditions would aid clinicians and researchers in diagnosing and prognosing myelin-associated neuropathies.
The spinal nerve roots can be damaged as they traverse the spinal canal, but are especially vulnerable in the intervertebral foramen, where the spinal nerve roots join to form the spinal nerves. Syndromes such as cervical radiculopathy, sciatica, intervertebral disc herniation, and root compression are caused by compression primarily from tumors or other lesions, which usually present with back or neck pain. Back or neck pain may be caused by a variety of musculoskeletal mechanisms and the physician needs to be able to examine the nervous system to determine if there is compression of nerve roots or the spinal cord. The ability to image and identify the source of chronic neck or back pain could enable surgeons to effectively treat these syndromes.
Myelin-labeling methodologies do exist, including the use of commercially available FluoroMyelin dyes for identification of high myelin content tissues. However, except for a few dyes such as bis-styrene-arylene dyes such as 1,4-bis(p-aminostyryl)-2-methoxy benzene (BMB), and (E,E)-1,4-bis(4′-aminostyryl)-2-dimethoxy-benzene (BDB), most of the publicly-disclosed dyes are unable to cross the blood nerve or blood brain barrier.
Myelin is a protein and lipid-rich matrix formed by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). Because two different cell types in CNS and PNS produce myelin, namely oligodendrocytes and Schwann cells respectively, there are similarities and differences in protein and lipid composition depending on the source of myelin. In both instances, myelin is composed of about 80% lipid fraction and about 20% protein fraction. Numerous studies have examined the molecular components of both fractions.
The lipid fraction in myelin contain cholesterol, cholesterol ester, cerebroside, sulfatide, sphingomyelin, phosphotidylethanolomine, phosphotidylcholine, phosphotidylserine, phosphotidylinositol, triacylglycerol, and diacylglycerol. The protein fraction is composed of several proteins, which include myelin basic protein (MBP), peripheral myelin protein 22 (PMP22), connexin 32 and myelin-associated glycoprotein (MAG), which are, produced by both PNS and CNS cells; the protein myelin protein zero (MPZ), produced by the PNS only; and proteolipid protein, produced by CNS cells only.
MBP is a major protein component of myelin at 5%-15%, which translates into about 5 mM concentration of MBP. Techniques such as circular dichroism, NMR and EPR spectroscopy, atomic force microscopy and others, suggest that MBP may have a compact C-shaped form with a core element of beta-sheet structure, but only when associated with lipids. The interaction of myelin basic protein to lipids can cause conformational variability and may be critical for function.
An agent that selectively binds to MBP may result in improvements in myelin staining and thereby aid in nerve visualization. Nerve visualization my be further improved through, optimal elimination of unbound and nonspecifically bound dye, and improved optical properties to allow enhanced contrast between myelin and surrounding tissue. Optical properties in the near infrared range (NIR), between 700-900 nm, are ideal for visualization of myelin in vivo. In the NIR range the absorption of water, hemoglobin, and lipid are minimal, and scatter is reduced such that photon penetration is improved. Also, autofluorescence is low and the NIR light penetrates deep into tissue and is less affected by scatter.